Expression Of MiRNAs And Genes Coding For The Enzymes Involved In MiRNA Biogenesis In Mouse Preimplantation Embryos

MicroRNAs (miRNAs) are a newly found 19-25nt single stranded noncoding RNAs, and were distributed widely in Eukaryotes from C. elegans to human beings. MiRNAs have a characteristic of highly evolutionarily conservation and exihibit tissue-specific or developmental-stage-specific expression.Most of their genes are not randomly distributed but often closely clustered on the genome. MiRNAs act as a gene regulation molecular and function through the regulation of target genes by imperfect or perfect base-pairing with their target mRNAs, then either lead to the degradation of the target transcript (near perfect complementarity), or the inhibition of the protein translation.Through above mechnisim miRNAs play a important role in gene regulation network and play critical roles in many biological processes including the regulation of development, cell proliferation or differentiation, apoptosis and cancer formation.Since the important roles of lin-4 and let-7 in the development of nematodes were found, many studys reported that miRNAs and genes coding for the enzymes involved in miRNA biogenesis were expressed and have influence on the development of post implantation embryos. But presently there is no report about expression and function of miRNAs in mouse preimplantation embryos. In this study, we intended to investigate the expression of miRNA and genes coding for the enzymes involved in miRNA biogenesis to find the evidence of miRNA acting on development and differentiation of preimplantation embryosAIM: To set up a system about murine superovulation and mating and to establish a platform of embryo recovery to investigate the expression of miRNAs in mouse preimplantation embryos and to futher illustrate the possible role and significance of miRNA in the preimplantational embryonic development.METHODS AND RESULTS:1. We used a method of miRNA amplification system combined with miRNA microarry technique to analyze miRNA expression across mouse oocyte and preimplantation embryos. For the first time, we found that fifty-five, fifty-three, sixty-two and seventy-two miRNAs were expressed respectively in mouse oocytes, 1.5dpc(2cell), 2.5dpc(4-8cell) and 4.5dpc(blastocyst) embryos. Ninty-four miRNAs were expressed in preimplantation embryos and thirty-two were expressed commonly in each stage of development and sixty-two were expressed in the four different stages of development. Of the commonly expressed 32 miRNAs, 16 showed high levels. Of the 62 stage-specific miRNAs, cluster of miR-290~295 were expressed only in blastocyst, which were previously cloned from murine ES cells and were considered to have ES cell-specific functions. Of the identified miRNAs in microchip, the roles of miR-155 and miR-181 were clearly. Through analyzing the function of the two miRNAs suggests that miRNAs might play a role in maintenance of the embryonic cell differentiation and in the regulation of early mammalian development.2. We used qRT-PCR to study the expression level of miR-721 and let-7e across mouse oocyte, 4-8cell and blastocyst embryos. The results of qRT-PCR show that both of them were expressed and the level of expression decreases with the development.3. We used RT-PCR to investigate the expression of Dicer and DGCR8 across mouse oocytes, 1.5dpc (2cell), 2.5dpc (4-8cell), 3.5dpc (morula) and 4.5 dpc(blastocyst) embryos. The results indicate that the two genes are all expressed across oocyte and preimplantation embryos.They are the important factor involved in miRNA biogenesis of preimplantation embryos and might play a role in embryo development.CONCLUSION: Through successfully setting up the system of murine ovulation and mating and the platform of embryo recovery, we identified miRNAs and genes of Dicer and DGCR8 in mouse oocyte and preimplantation embryos. In addition, we analyzed the function of the expressed miRNAs and genes coding for the enzymes involved in miRNA biogenesis. Our results suggest that miRNAs might play a role in the development and differentiation of the embryonic cells. Our analyses also indicate that there might be a correlation between miRNA and genomic activation after 2-cell development in mouse embryo.